Diabetes mellitus is a leading cause of death and affects over 20 million people in North America alone and about 200 million worldwide (American Diabetes Association: www.diabetes.org and Canadian Diabetes Association: www.diabetes.ca; World Health Organization: http://www.who.int/diabetes). The two major forms of the disease are type I and type II diabetes. Both are characterized by a progressive decrease in beta-cell mass and beta-cell function.
Type I diabetes (also called juvenile diabetes) is a complex T-cell dependent autoimmune disease (Juneja and Palmer, Autoimmunity. 1999; 29(1):65-83) that typically develops at a young age. Type I diabetes results from the autoimmune destruction of islet beta-cells with consequent insulin deficiency and dependence on exogenous insulin treatment. The focal infiltration of the endocrine pancreas by mononuclear cells and a strikingly decreased functional beta-cell mass constitute the histopathological hallmarks of the disease at diagnosis, but there is a marked inter-individual variability in terms of the extent of these lesions. The beta-cell apoptosis occurs as a result of autoimmune destruction involving T cell infiltration of the islets of Langerhans (Lee et al., Mol Genet Metab. 2004; 83(1-2):82-92; Mandrup-Poulsen, Biochem Pharmacol. 2003; 66(8):1433-1440; Sesti, Ann Med. 2002; 34(6):444-450; Mathis et al., Nature. 2001; 414(6865):792-798). To study the underlying molecular mechanism of diabetes, animal models have been developed. For example, streptozotocin (STZ)-induced insulin-deficient rats or mice mimic the T-cell mediated inflammation and destruction of islet beta-cells seen in diabetes patients. The non-obese diabetic (NOD) mouse is another model of autoimmune diabetes where islet-antigen reactive T cells infiltrate the islets of Langerhans and kill islet beta cells, and/or initiate an inflammatory process that results in islet beta cell death (Anderson and Bluestone, Annu Rev Immunol. 2005; 23:447-485).
Insulin therapy is a major intervention for the treatment of type I diabetes. Pancreatic islet transplantation is an effective therapy (Shapiro et al., N Engl J. Med. 2000; 343(4):230-238) but is limited largely by the limited resources of human islet. In addition, immune-suppressors need to be used in the islets-transplanted patients for life. Though insulin therapy is used for most patients with type I diabetes, insulin is not a cure as it is difficult to maintain blood glucose levels within a narrow physiological range, and it does not prevent the progression of the disease nor the development of severe diabetic complications.
Type II diabetes is a polygenic disorder typically diagnosed in adulthood and is characterized by three major abnormalities that contribute to the development of hyperglycemia: 1) peripheral insulin resistance, 2) excessive hepatic glucose production, and 3) pancreatic beta-cell dysfunction. Insulin resistance is defined as the reduced response to insulin in peripheral tissues, mainly the skeletal muscle cells, leading to impaired glucose transport into these tissues (Kahn and Goldfine, J Diabetes Complications. 1993; 7(2):92-105; Weyer et al., J Clin Invest. 1999; 104(6):787-794). Insulin resistance can also occur in the liver where insulin is unable to efficiently suppress hepatic glucose production (Kahn and Goldfine, 3 Diabetes Complications. 1993; 7(2):92-105; Lam et al., Am J Physiol Endocrinol Metab. 2002; 283(4):E682-E691). Furthermore, excessive pancreatic glucagon secretion is also a major contributor to the disproportionate over-production of hepatic glucose (Unger and Orci, Arch Intern Med. 1977; 137(4):482-491). As a result of insulin resistance, the body's demand for insulin is increased. In the early stages of insulin resistance, the blood glucose levels can still be maintained within a normal range via a compensatory mechanism increasing insulin output that involves increased beta-cell mass in the pancreas (Bonner-Weir, Trends Endocrinol Metab. 2000; 11(9):375-378; Bonner-Weir, Endocrinology. 2000; 141(6):1926-1929). Numerous studies indicate that insulin resistance on its own is not sufficient to trigger the onset of diabetes, if the beta-cell compensatory capacity is maintained (Weyer et al., Diabetes. 1999; 48(11):2197-2203). However, in the long term and when insulin resistance becomes severe, the increased demand for insulin leads to beta-cell exhaustion, decreased insulin production, and the development of fasting hyperglycaemia and overt diabetes (DeFronzo, Diabetes. 1988; 37(6):667-687; Kahn et al., J Nutr. 2001; 131(2):354S-360S; Weyer et al., J Clin Invest. 1999; 104(6):787-794). The obese insulin resistance db/db mouse is a severe animal model of type II diabetes. These mice are deficient in leptin signaling (Herberg and Coleman, Metabolism. 1977; 26(1):59-99; Chen et al., Cell. 1996; 84(3):491-495).
Conventional treatments for type II diabetes include diet and exercise as well as pharmacological interventions with sulphonylureas, metformin and insulin. These treatments generally fail to prevent the long-term decline in glycemic control and the beta-cell dysfunction in most patients (Matthews et al., Diabet Med. 1998; 15(4):297-303; Turner et al., JAMA. 1999; 281(21):2005-2012). Clinical management of type II diabetes using stepwise approaches also eventually fails to sustain glycemic control where, for most patients, there is a an unavoidable progression from diet and exercise to pharmacotherapy with a single agent, to combination therapy and finally to insulin (Turner et al., JAMA. 1999; 281(21):2005-2012; Gerich, Eur J Clin Invest. 2002; 32 Suppl 3:46-53). The ineffectiveness of these therapies in preventing either the progression of type II diabetes or the long-term complications associated with this disease may be a consequence of the focus of these approaches on the symptoms (i.e. hyperglycemia) rather than the cause of type II diabetes (Gerich, Eur J Clin Invest. 2002; 32 Suppl 3:46-53).
Glucagon-like peptide-1 (7-36)-amide (GLP-1) is an insulinotropic hormone (Brubaker and Drucker, Endocrinology. 2004; 145(6):2653-2659; Perfetti and Merkel, Eur J Endocrinol. 2000; 143(6):717-725; Hoist, Gastroenterology. 1994; 107(6):1848-1855; Holst and Gromada, Am J Physiol Endocrinol Metab. 2004; 287(2):E199-E206) that is secreted from intestinal L-cells in response to nutrient ingestion and promotes nutrient absorption via regulation of islet hormone secretion (Drucker, Diabetes. 1998; 47(2):159-169). GLP-1 binds to the GLP-1 receptor (GLP-1R), a G-protein coupled receptor (GPCR). GLP-1R is expressed mainly by pancreatic beta-cells, and to some extent by cells of other tissues (lungs, heart, kidney, GI tract and brain), and is coupled to the cyclic AMP (cAMP) second messenger pathway to initiate its biological actions (Drucker, Endocrinology. 2001; 142(2):521-527; Brubaker and Drucker, Endocrinology. 2004; 145(6):2653-2659), (Brubaker and Drucker, Receptors Channels. 2002; 8(3-4):179-188; Brubaker and Drucker, Endocrinology. 2004; 145(6):2653-2659; Thorens, Proc Natl Acad Sci USA. 1992; 89(18):8641-8645) protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs) (Miura and Matsui, Toxicol Appl Pharmacol. 2006; Holz, Horm Metab Res. 2004; 36(11-12):787-794). Activation of other protein kinases including Akt (protein kinase B) and MAPK (Mitogen-Activated Protein Kinases (MAPK)) (Brubaker and Drucker, Endocrinology. 2004; 145(6):2653-2659; Wang and Brubaker, Diabetologia. 2002; 45(9):1263-1273; Wang et al., Diabetologia. 2004; 47(3):478-487) is also found to be important in mediating GLP-1 action in promoting beta-cell growth and inhibiting apoptosis.
GLP-1 enhances pancreatic islet beta-cell proliferation and inhibits beta-cell apoptosis in a glucose-dependent fashion (Nauck et al., Horm Metab Res. 1997; 29(9):411-416; Nauck, Horm Metab Res. 2004; 36(11-12):852-858; Drucker, Diabetes. 1998; 47(2):159-169). GLP-1 also augments insulin secretion and lowers blood glucose in rodents as well as in humans in both type I diabetes (Gutniak et al., Diabetes Care. 1994; 17(9):1039-1044) and type II diabetes (Nauck et al., Diabetes. 1997; 105(4):187-195; Todd et al., Eur J Clin Invest. 1997; 27(6):533-536; Nathan et al., Diabetes Care. 1992; 15(2):270-276). In animals models of type II diabetes, GLP-1 or its long-acting potent analogue exendin-4 (Ex4) treatment prevented onset of diabetes (Wang and Brubaker, Diabetologia. 2002; 45(9):1263-1273; Tourrel et al., Diabetes. 2002; 51(5):1443-1452) by enhancing beta-cell growth and inhibiting apoptosis (Wang and Brubaker, Diabetologia. 2002; 45(9):1263-1273; Wang, Endocrinology Rounds. 2004; 3(7); Wang et al., Diabetologia. 2004; 47(3):478-487; Tourrel et al., Diabetes. 2002; 51(5):1443-1452). GLP-1 has demonstrated clinical efficacy in type II diabetes (Meier and Nauck, Diabetes Metab Res Rev. 2005; 21(2):91-117). Studies demonstrated that in insulin-secreting beta-cells, the apoptosis and necrosis induced by cytokines could be significantly blocked by GLP-1 or exendin-4 (Ex4) (Saldeen, Endocrinology. 2000; 141(6):2003-2010; Li et al., Diabetologia. 2005). Treatment with GLP-1/Ex4 stimulated beta-cell neogenesis in STZ-treated newborn rats resulting in persistently improved glucose homeostasis at an adult age (Tourrel et al., Diabetes. 2001; 50(7):1562-1570). Furthermore, administration of GLP-1/Ex4, combined with immunosuppression by polyclonal anti-T cell antibody, induced remission in 88% of diabetic NOD mice (Ogawa et al., Diabetes. 2004; 53(7):1700-1705).
U.S. Pat. No. 6,899,883 and U.S. Pat. No. 6,989,148 disclose methods of treating type I diabetes using insulin and glucagon-like peptide 1(7-37) or glucagon-like peptide 1(7-36) amide. Native GLP-1 has a short circulating half-life (t1/2<2 min) that results mainly from rapid enzymatic inactivation including dipeptidyl-peptidase IV (DPP-IV) (Drucker, Diabetes. 1998; 47(2):159-169), and/or renal clearance (Montrose-Rafizadeh et al., Endocrinology. 1999; 140(3):1132-1140). Therefore, continuous subcutaneous infusion by pump is necessary to maintain GLP-1 action in vivo (Toft-Nielsen et al., Diabetes Care. 1999; 22(7):1137-1143). A DPPIV inhibitor can increase the half-life of GLP-1, DPPIV also inactivates several other peptide hormones and some chemokines (Meier and Nauck, Diabetes Metab Res Rev. 2005; 21(2):91-117), and its inhibition may lead to adverse reactions. In this respect, efforts have been made to develop pharmaceutical long-acting degradation-resistant GLP-1 mimetic peptides. Human GLP-1 analogues with amino acid substitutions (Ahren and Schmitz, Horm Metab Res. 2004; 36(11-12):867-876; Green et al., Curr Pharm Des. 2004; 10(29):3651-3662) and/or N-terminal modifications including fatty acylated (Chang et al., Diabetes. 2003; 52(7):1786-1791) and N-acetylated (Liu et al., Cell Biol Int. 2004; 28(1):69-73) modifications exhibit prolonged circulating t1/2, and potently reduce glycemic excursion in diabetic subjects (Chang et al., Diabetes. 2003; 52(7):1786-1791). Ex4, a reptilian peptide with high sequence homology to mammalian GLP-1 is a potent GLP-1R agonist (Fineman et al., Diabetes Care. 2003; 26(8):2370-2377). Furthermore, albumin protein-conjugated GLP-1 (Albugon) also has the anti-diabetic and other beneficial activities of GLP-1 along with a prolonged half-life (Kim et al., Diabetes. 2003; 52(3):751-759).
Although DPP-IV-resistant GLP-1R agonists as well as Ex4 appear to be promising therapeutic drug candidates for the treatment of diabetes, these peptides require once- or twice-daily injections and/or combination therapies with oral diabetic medications. The substantially prolonged half-life of GLP-1-albumin fusion proteins, or GLP-1 fusion IgG4 fusion proteins such as those described in WO 02/46227 or WO 05/000892, is likely the result of reduced renal clearance due to the larger size. However, in vitro studies have shown that a fusion protein displays a lower potency (Kim et al., Diabetes. 2003; 52(3):751-759). This has fostered complementary efforts to generate more potent longer-acting agents with sustained efficacy in vivo.
Thus, there still remains a need to develop effective treatment strategies that target the molecular mechanisms underlying type I and type II diabetes rather than the consequences. Intervention with therapies that target both the beta-cell dysfunction and insulin resistance are desirable. Therefore, a therapy that promotes beta-cell growth and also protects from beta-cell death is necessary for effective treatment of this disease.